Recurrent prostate cancer is often characterized by upregulation of the anti-apoptotic protein Bcl-2 and resistance to conventional therapies. Bcl-2 is a major target of photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 in human prostate cancer cells. Photodamage is readily detected on western blots as loss of the 26-kDa proteins and gain of multiple higher-molecular-weight bands of Bcl-2 complexed to various proteins. The proposed research will characterize the role of photodamage to Bcl-2 in androgen-sensitive and -independent prostate cancer cell lines. The primary hypothesis is: (a) Bcl-2 in mitochondria and other cytoplasmic membranes is a key target of PDT in prostate cancer cells; (b) the initial PDT lesions cause Bcl-2 to complex with neighboring molecules; and (c) photodamage to Bcl-2 interferes with its anti-apoptotic functions. In Specific Aim 1, we will evaluate the relationship between membrane localization and binding of Bcl-2 and the sensitivity of the protein to photodamage by Pc 4-PDT in prostate cancer cells. Bcl-2 mutated in the critical a5/a6 domain or deleted in regions necessary for interaction with pro-apoptotic molecules will be studied by transient and stable expression in prostate cancer cells, in comparison to wild-type Bcl-2. For Specific Aim 2, we will isolate membrane components complexed to Bcl- 2 in prostate cancer cells using immunoprecipitation or affinity chromatography and identify proteins by mass spectrometry. Immunoprecipitation and western blotting will identify other proteins. In Specific Aim 3, we will evaluate the role of Bcl-2 photodamage in determining the fate of prostate cancer cells. The induction of apoptosis, interference with key steps in apoptosis, and the loss of clonogenicity will be studied in prostate cancer cells overexpressing Bcl-2 or selected mutants. Inducers will include Pc 4-PDT, a chemical ligand of Bcl-2, or other oxidative stresses. Treatment options for prostate cancer patients are limited, partly due to the relatively poor sensitivity of recurrent tumors to conventional therapies. The proposed investigation will elucidate the cellular mechanism of a relatively new modality for prostate cancer that may convert the cells from an apoptosis-resistant to an apoptosis-sensitive state through photodamage to Bcl-2.